Cholesterol Promotes Colorectal Cancer Growth by Activating the PI3K/AKT Pathway

Globally, the incidence of colorectal cancer (CRC) increases each year, with an unhealthy diet representing one of the major pathogenic risk factors for CRC. Cholesterol is a vital dietary ingredient required to maintain the normal function of the body; however, disturbances in cholesterol levels have been discovered to exert a significant role in tumorigenesis. The present study is aimed at investigating the role of cholesterol in the occurrence of CRC. Briefly, CRC model mice were established through an intraperitoneal injection of azoxyemethane (AOM) and were subsequently either fed a normal diet (ND), high-fat diet (HFD), or high-fat high-cholesterol diet (HFHC). Furthermore, in vitro experiments were performed following the treatment of SW480 and HCT116 cells with cholesterol, and the cell viability and colony formation rate of CRC cells were analyzed. The findings identified that cholesterol levels were increased in CRC tissues compared with adjacent normal tissues. In contrast, the serum levels of cholesterol were decreased in patients with CRC compared with the healthy controls; however, no significant differences were observed in the cholesterol levels between stage I + II and stage III + IV patients with CRC. Notably, CRC model mice fed with an HFD or HFHC recorded a larger body weight compared with those mice fed a ND; however, no significant differences were reported in the number of tumors formed in each group. Furthermore, the tumor size in the HFHC group was discovered to be increased compared with the ND and HFD groups, and HGD and the pathological morphology were the most pronounced in the HFHC group. Moreover, mice in the HFHC group presented the highest ratio of Ki-67-positive staining and the lowest ratio of TUNEL-positive staining compared with those in the two other groups. Cholesterol treatment also increased the cell viability and clonality of SW480 and HCT116 cells. In addition, the protein expression levels of phosphorylated-AKT were upregulated in cholesterol-induced CRC cells and tissues, whereas the treatment with BAY80-6946 attenuated the cholesterol-induced increases in the cell viability, colony formation ability, and tumor size. In conclusion, the findings of the present study suggested that cholesterol may stimulate the progression of CRC by activating the PI3K/AKT signaling pathway; however, cholesterol may not affect the number of tumors formed in CRC. In addition, cholesterol was discovered to mainly affect the advanced stages of CRC rather than the early stages.


Introduction
It is currently estimated that~90% of colorectal cancer (CRC) cases are sporadic, while the remaining 10% of cases are classified as hereditary CRC [1,2]. However, interestingly, previous studies have uncovered the crucial function of the intestinal flora in the progression of CRC [3].
In-depth studies of the pathogenesis of CRC have identi-fied that its occurrence is closely associated with lifestyle factors; for example, both smoking and alcohol overdose increased the susceptibility to CRC. Therefore, dietary factors must not be overlooked since these factors are also related to our lifestyles; for instance, red meat has been discovered to significantly increase the risk of CRC development due to the heme it contains, which increases the accumulation of toxins in the intestine [4]. Thus, it has been suggested that increased vegetable intake may reduce the risk of CRC arising from high red meat consumption [5].
A previous in vivo study demonstrated that a high cholesterol diet significantly increased the number of CRC tumors formed in APC Min/+ mice [6,7]. Cholesterol is a type of fat, which is found within numerous types of food, especially processed foods, including red meat, seafood, and sausages. Cholesterol is necessary to maintain the normal function of the body [8,9].
In our current research, CRC model mice were established by azoxymethane (AOM) administration and subsequently either fed a normal diet (ND), high-fat (HFD), or high-fat high-cholesterol (HFHC) diet. Our present research aimed to provide a novel target for the better understanding of CRC.

Materials and Methods
2.1. Cell Culture. Based on the calculated IC 50 values of BAY80-6946 in SW480 and HCT116 cells, the cells were treated with 11.2 or14.6 μM BAY80-6946 for the following experiments, with the medium being replaced every three days.

Clinical Samples.
The tumor tissues and the paracarcinoma tissues were collected from the enrolled patients in our hospital. Serum levels of cholesterol in the patients with CRC and healthy controls were recorded for analysis, and the characteristics of the patients with CRC are summarized in Table 1. The Ethics Committee of Chinese PLA General Hospital approved our current study (no: S2019-032-02). Written informed consent was obtained from each patient and/or the guardians.

Animal Experiments.
Male C57/BL6 mice (age, 6 weeks) were assigned into three groups (10 mice/group): (i) a ND group; (ii) a HFD group, in which the pellet fed to mice in the ND group was mixed with 10% lard and 10% egg yolk; and (iii) a HFHC group, in which the pellet fed to mice in the ND group was mixed with 10% lard, 10% egg yolk, and 1% cholesterol [10]. The mice were injected at 6-weeks old with 10 mg/kg AOM six times weekly and sacrificed at week 24 following the first AOM injection.
For mechanistic studies, the mice were assigned into four groups (6 mice/group): (i) a ND group; (ii) a HFD group; (iii) a HFHC group; and (iv) a HFHC + BAY group, in which mice were injected with BAY80-6946 (HY-5346; MedChemExpress). AOM administration was the same as previously described. During the experimental period, the mouse body weight was regularly recorded. The present study was approved by the Animal Ethics Committee of Chinese PLA General Hospital Animal Center (no. [2019]-031).

MTT Assay.
Following the cell incubation, 20 μl MTT solution was added to each well for incubation for 4 h. Then, the mixture was shaken at a low speed for 10 min to dissolve the crystals sufficiently. The absorbance was determined at a wavelength of 570 nm using an enzyme-linked immunosorbent detector to calculate the IC 50 values. The experiment was repeated in triplicate.
2.6. Colony Formation Assay. Colony formation assay was performed according to the manufacturer's protocols. Then, cells were fixed for 1 h in methanol and stained with 0.1% violet crystal for 20 min. After removing the staining solution, the stained colonies were air-dried and visualized under a microscope.

2.7.
Ki-67 and TUNEL Staining. CRC tissues were harvested from the mice in each group, and Ki-67 (cat. no. GTX20833; GeneTex, Inc.) and TUNEL staining (cat. no. ab206386; Abcam) were performed using their commercial kits. The positive expression in cells was subsequently calculated.
2.8. Determination of Cholesterol Levels. Cholesterol levels were determined using 2 mg CRC or normal tissues using a cholesterol quantification kit (cat. no. ab65359; Abcam). Briefly, to determine the serum levels of cholesterol, 50 μl serum samples were collected and diluted twice in PBS.

Statistical
Analysis. SPSS version 20.0 software was used to perform the statistical analysis. Comparisons between groups were analyzed using Student's t-test or one-way Journal of Oncology ANOVA, followed by LSD. P < 0:05 was considered to be statistically significant.

Cholesterol Levels Are Increased in CRC.
The levels of cholesterol were significantly higher in CRC tissues compared with that in the normal tissues (Figure 1(a)). On the contrary, the serum levels of cholesterol were decreased in 36 CRC samples (Figure 1(b)) [10]. Additionally, the cholesterol levels were compared in patients with CRC with different tumor stages; however, no significance was identified in the cholesterol levels between stage I + II and stage III + IV patients with CRC (Figure 1(c)), which may be explained

HFHC Diet Stimulates the Growth of CRC.
To investigate the effects of cholesterol in CRC, mice were intraperitoneally injected with AOM once a week for 6 consecutive weeks, while being fed different diets during the experiment (Figure 2(a)). At week 24, the mice were sacrificed for the following experiments. The body weight in ND group was markedly reduced than the HFD and HFHC groups (Figure 2(b)). In addition, the number of CRC tumors formed was similar in each group, with no significant differences found. Nevertheless, the tumor size in the HFD and HFHC groups was increased compared with the ND group (Figure 2(c)). The percentage of HGD remained the highest in HFHC group. Meanwhile, the histopathological examination confirmed the occurrence of CRC in mice (Figure 2(d)). Moreover, the level of cholesterol was discovered to be with a positive correlation with tumor size in CRC patients (r = 0:4486; P = 0:0025; Figure 3). These findings suggested that cholesterol may stimulate the growth of CRC; however, it failed to increase the tumor number.

Cholesterol Stimulates Proliferation and Inhibits
Apoptosis in CRC. In CRC tissues harvested from mice in the HFHC group, the cholesterol levels were increased compared with the two other groups (Figure 4(a)). In addition, both the cell viability and colony forming abilities were increased in SW480 and HCT116 cells following cholesterol induction (Figure 4(b)). Subsequently, the tumor tissues of CRC mice were subjected to Ki-67 and TUNEL staining; as the pathological results demonstrated, Ki-67-positive staining was the most pronounced in HFHC group, further confirming the cholesterol-induced proliferative stimulation in CRC (Figure 4(c), upper lane). Moreover, the TUNELpositive cell ratio was the highest in ND group, suggesting that cholesterol treatment may suppress apoptosis in CRC (Figure 4(c), bottom lane).

Cholesterol Stimulates Development of CRC by
Regulating PI3K/AKT. Recently, squalene epoxidase (SQLE), a crucial rate-limiting enzyme in cholesterol synthesis, was proven to enhance the development of liver cancer [11]. Cholesterol-induced SW480 and HCT116 cells exhibited upregulated p-AKT, and a similar trend was observed in the CRC tissues of the HFHC group, whereas AKT expression levels remained unchanged ( Figure 5(a)). Relevant previous studies have also illustrated the roles of MAPK (p38) and STAT3 in enhancing the growth of CRC and breast cancer [12,13]. Nevertheless, p-STAT3, STAT3, p-p38, and p38 levels were similar in all CRC cells, regardless of cholesterol treatment. In addition, in the CRC model mice in the ND, HFD, and HFHC groups ( Figure 5(b)).
The IC 50 values of BAY80-6946 in SW480 and HCT116 cells were determined to be 5.6 and 7.  Journal of Oncology a decreased cell viability and colony formation rate ( Figure 5(d)). Subsequently, CRC model mice were assigned to the ND, HFD, HFHC, and HFHC + BAY80 − 6946 groups (injection with BAY80-6946); however, no significant differences were identified in the body weight amongst the four groups ( Figure 5(e)). Nonetheless, following the injection with BAY80-6946, the tumor size decreased, while the number of tumors formed was unaltered ( Figure 5(f)). These findings indicated that cholesterol may stimulate CRC growth via activating the PI3K/ATK signaling pathway.

Discussion
An increasing evidence demonstrated the close association between lifestyle and the tumorigenesis of CRC. Processed foods provide convenience to daily life; however, they have also been discovered to increase the disease susceptibility.
The purpose of this current research was to explore the potential influence of cholesterol on the growth of CRC. It was suggested that the cholesterol levels were increased in CRC, suggesting the potential involvement of cholesterol in CRC development. These results were consistent with the   [14,15]. In addition, we also showed that the serum level of cholesterol in CRC patients was reduced, which was consistent with a previous study [10]. The ability of patients with CRC to absorb nutrients is poor. Meanwhile, the increased number of cholesterol-absorbed receptors and the reduction in cholesterol-depleting receptors has been discovered to result in the accumulation of cholesterol in cancer tissues, while decreasing the serum levels [16]. Our current study compared the levels of cholesterol in patients with CRC with different tumor stages; however, no significant differences were identified between the stages. This finding may be explained by the small sample size and variation between different individuals.
To further determine the relationship between cholesterol levels and CRC, in vivo CRC model mice were established. The results revealed that the HFHC diet resulted in a larger tumor size in the CRC mice compared with the mice fed a ND or HFD. These results suggested that cholesterol may not pose an impact on the initial stages of CRC, but it may influence tumor growth, which can be further suggested from the observation that the patient's tumor size was positively correlated with the cholesterol levels. Furthermore, we discovered that cholesterol enhanced proliferative ability and inhibited apoptosis in CRC, which are primary causes of increased tumor growth.
This current research was the first attempt to research the underlying function of cholesterol in cancer. We demonstrated that cholesterol upregulated p-AKT expression levels, whereas the PI3K inhibitor reversed the regulatory effect of cholesterol in CRC, further confirming the role of this pathway in CRC progression [17][18][19].
Cholesterol may be a risk factor for the onset of CRC [20]. Interestingly, the present study revealed that a highfat, but low-cholesterol diet, barely influenced the growth of CRC, which was inconsistent with the findings of a previous study [21]. However, there is an increasing proportion of obese individuals at present, and cholesterol levels in obese patients were reported to be markedly increased compared with in normal people [22]. Thus, all the above results demonstrated that obesity might be a risk factor for CRC.

Conclusions
In conclusion, it was suggested that cholesterol stimulated CRC development via activating PI3K/AKT; however, cholesterol was not found to affect the tumor number in CRC. Interestingly, this finding suggested that cholesterol may primarily affect the advanced stages of CRC rather than the early stages.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.